Abstract

BACKGROUND:

Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238-245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol.

METHODS AND FINDINGS:

Using human skin tissue, we report here the presence of specific [(3)H]-resveratrol binding sites (K(D) = 180 nM) that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3-3 mM) resulted in cell death which was reduced by resveratrol (EC(50) = 14.7 µM), and to a much lesser extent by the resveratrol analogue piceatannol (EC(50) = 95 µM) and epigallocatechin gallate (EC(50) = 200 µM), a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP.

CONCLUSION:

Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.

Photomicrographs of the autoradiographic distribution of [3H]-resveratrol binding sites in human skin.

Upper panel (A). Human skin sections were incubated during 8 months with [3H]-resveratrol (25, 60 and 130 nM), stained with hematoxylin/eosin and visualized using a microscope. Higher levels of silver grains were observed in the epidermis as compared to the dermis. Total and non-specific binding represents [3H]-resveratrol binding with or without cold resveratrol (100 µM), respectively. Lower panel (B). Photomicrographic distribution of human skin incubated with [3H]-resveratrol (25, 60 and 130 nM) and exposed to liquid emulsion for 8 months. The widespread distribution of silver grains (black dots) in the epidermis suggested that [3H]-resveratrol binding sites are mostly located in keratinocytes, which makes about 90% of skin epidermis. The number of silver grains is similar in undifferentiated (cells in the basal layer) and differentiated (cells at the surface of the skin) keratinocytes.

Competition binding profile of resveratrol against [3H]-resveratrol binding site in human skin and brain homogenates.

Brain homogenates and human skin sections were incubated with [3H]-resveratrol in the presence of increasing concentration of cold resveratrol (0.1 nM–100 µM). Each point represents the mean±SEM of data obtained from three to five determinations, each performed in triplicate and expressed as percentage of specific binding.

Binding experiments were performed at 4°C in the presence or the absence of 100 µM unlabelled resveratrol; specific (round points) binding represents the difference between total (square points) and non-specific (triangle points) binding. Insert shows the mathematical transformation as Scatchard plots of the binding isotherms of [3H]-resveratrol. Each point was done in triplicate and repeated 3 times.

Effect of resveratrol against cell death induced by SNP in HaCaT cells.

Cells were exposed to SNP (0.3–3 mM) in the presence or absence of resveratrol (1–30 µM). Cell viability was determined 24 hours later using both the MTT and calcein AM assays. Values represent mean ± SEM of at least three separate experiments. *p<0.05, **p<0.01 comp.

Effect of resveratrol against apoptotic events induced by SNP in HaCaT cells.

Cells were exposed to SNP (0.3–3 mM) in the presence or absence of resveratrol (1–30 µM). Cell viability was determined 24 hours later using both the SYTO 16 (A) and JC-1 (B) assays. Values represent mean ± SEM of at least three separate experiments. *p<0.05, **p<0.01 compared to groups treated with SNP alone. +p<0.05 compared to control group.